HCMV is a member of the betaherpesvirus family (Mocarski (1996) Cytomegaloviruses and Their Replication Third ed., vol. 2. Lippincott—Raven Publishers, Philadelphia; Roizman (1996) Herpesviridae Third ed., vol. 2. Lippincott—Raven Publishers, Philadelphia). Other members of this family include human herpesvirus 6 (HHV-6), and human herpesvirus 7 (HHV-7), and all are widely distributed in human populations. During productive replication, the 230 kilobase pair (kbp) double stranded DNA viral genome replicates by a rolling circle mechanism, which generates long head-to-tail concatemers that are cleaved to unit length and packaged in capsids. The state of the HCMV genome during latency remains unidentified and is likely to be circular and extrachromosomal (Bolovan-Fritts et al. (1999) Peripheral blood CD14(+) cells from healthy subjects carry a circular conformation of latent cytomegalovirus genome Blood 93:394-8). The HCMV genome has been detected in cells within the hematopoietic lineage as early as CD34+ progenitors and up through differentiated macrophages (Hahn et al. (1998) Cytomegalovirus remains latent in a common precursor of dendritic and myeloid cells Proc Natl Acad Sci USA 95:3937-42; Kondo et al. (1994) Human cytomegalovirus latent infection of granulocyte-macrophage progenitors Proc Natl Acad Sci USA 91:11879-83; Mendelson et al. (1996) Detection of endogenous human cytomegalovirus in CD34+ bone marrow progenitors J Gen Virol 77:3099-102; Slobedman & Mocarski (1999) Quantitative analysis of latent human cytomegalovirus J Virol 73:4806-12).
Defective HSV viruses created by high multiplicity serial passage of virus stocks have been described on numerous occasions and have been characterized in detail at the molecular level (Cuifo & Hayward (1981) Tandem repeat defective DNA from the L segment of the HSV genome Martinus Nijhoff, The Hague; Frenkel et al. (1976) Anatomy of herpes simplex virus DNA VI. Defective DNA originates from the S component J Virol 20:527-31; Locker & Frenkel (1979) Structure and origin of defective genomes contained in serially passaged herpes simplex type 1 J Virol 29:1065-1077; Murray et al. (1975) Cyclic appearance of defective interfering particles of herpes simplex virus and the concomitant accumulation of early polypeptide VP175, Intervirology 5:173-84; Schroder et al. (1975) An unusual defective genotype derived from herpes simplex virus strain ANG Intervirology 6:270-84; Vlazny & Frenkel (1981) Replication of herpes simplex virus DNA: localization of replication recognition signals within defective virus genomes Proc Natl Acad Sci USA 78:742-667). Naturally occurring HSV defective viruses and laboratory derived HSV amplicon vectors are composed of head-to-tail tandem reiterations of subgenomic regions containing a functional origin of DNA replication (OriS or OriL) and a DNA cleavage/packaging signal (Barnett et al. (1983) Class I defective herpes simplex virus DNA as a molecular cloning vehicle in eucaryotic cells J Virol 48:384-95; Bearet al. (1984) Analysis of two potential shuttle vectors containing herpes simplex virus defective DNA J Mol Appl Genet. 2:471-84; Kwong & Frenkel (1995) Biology of herpes simplex virus (HSV) defective viruses and development of the amplicon system Viral Vectors p. 25-42, Academic Press, Inc.; Spaete & Frenkel (1982) The herpes simplex virus amplicon: A new eucaryotic defective-virus cloning-amplifying vector Cell 30:295-304; Stow (1982) Localization of an origin of DNA replication within the TRS/IRS repeated region of the herpes simplex virus type 1 genome Embo J 1:863-7; Stow & McMonagle (1983) Characterization of the TRS/IRS origin of DNA replication of herpes simplex virus type 1 Virology 130:427-38; Stow et al. (1983) Fragments from both termini of the herpes simplex virus type 1 genome contain signals required for the encapsidation of viral DNA Nucleic Acids Res 11:8205-20). These two cis-acting functions can be relatively small, ranging from about 90-150 base pairs (bp) for the ori and about 250-300 bp for the a sequence.
In contrast to HSV, HCMV does not efficiently produce defective virus genomes. This difference may be related to the distinct biology of the two viruses (Pari & Anders (1993) Eleven loci encoding trans-acting factors are required for transient complementation of human cytomegalovirus oriLyt-dependent DNA replication J Virol 67:6979-88). Only two reports have described the identification of what may potentially be HCMV defective viruses created by serial high multiplicity passage (Ramirez et al. (1979) Defective virions of human cytomegalovirus Virology 96:311-4; Stinski et al. (1979) DNA of human cytomegalovirus: size heterogeneity and defectiveness resulting from serial undiluted passage J Virol 31:231-9). These reports characterized HCMV defectives as very large subgenomic DNA molecules, in some cases extending over two thirds of the genome. The extent of these deletions has made it difficult to predict the minimal genetic complexity required for replication and packaging of an amplicon vector in a CMV infected cell.
The functional HCMV oriLyt is much more complex than either of the HSV oris; the HCMV oriLyt consists of multiple direct and inverted repeats and extends over at least 1500 bp (Anders et al. (1992) Boundaries and structure of human cytomegalovirus oriLyt, a complex origin for lytic-phase DNA replication Journal of Virology 66:3373-3384; Anders & Punturieri (1991) Multicomponent origin of cytomegalovirus lytic-phase DNA replication J Virol 65:931-937; Hamzeh et al. (1990) Identification of the lytic origin of DNA replication in human cytomegalovirus by a novel approach utilizing ganciclovir-induced chain termination J Virol 64:6184-6195; Masse et al. (1992) Human cytomegalovirus origin of DNA replication (oriLyt) resides within a highly complex repetitive region Proc Natl Acad Sci USA 89:5246-5250). HCMV is unique among the herpesviruses in not having an origin binding protein homolog that is required for DNA replication (Pari & Anders (1993) Eleven loci encoding trans-acting factors are required for transient complementation of human cytomegalovirus oriLyt-dependent DNA replication J Virol 67:6979-88). The HCMV a sequence varies in size from about 550 bp to 762 bp, and includes the conserved pac-1 and pac-2 cis-elements which determine the sites for cleavage of replicated viral DNA (Deiss et al. (1986) Functional domains within the a sequence involved in the cleavage-packaging of herpes simplex virus DNA J Virol 59:605-18; Kemble & Mocarski (1989) A host cellprotein binds to a highly conserved sequence element (pac-2) with the cytomegalovirus a sequence J Virol 63:4715-4728; Spaete & Mocarski (1985) The a sequence of the cytomegalovirus genome functions as a cleavage/packaging signal for herpes simplex virus defective genomes J Virol 54:817-824; Tamashiro et al. (1984) Structure of the heterogeneous L-S junction region of human cytomegalovirus strain AD169 DNA J Virol 52:541-8; Tamashiro & Spector (1986) Terminal structure and heterogeneity in human cytomegalovirus strain AD169 J Virol 59:591-604).
The infectivity of CD34+ cells from seropositive and seronegative subjects with HCMV has been tested both in vivo and in vitro (Sindre et al. (1996) Human cytomegalovirus suppression of and latency in early hematopoietic progenitor cells Blood 88:4526-33), and HCMV has been shown to infect cells of the hematopoietic lineage (Maciejewski et al. (1992) Infection of hematopoietic progenitor cells by human cytomegalovirus Blood 80:170-8; Mendelson et al. (1996) Detection of endogenous human cytomegalovirus in CD34+ bone marrow progenitors J Gen Virol 77:3099-102; Mocarski et al. (1993) Human cytomegalovirus in a SCID-hu mouse: thymic epithelial cells are prominent targets of viral replication Proc Natl Acad Sci USA 90:104-8; Soderberg et al. (1993) Definition of a subset of human peripheral blood mononuclear cells that are permissive to human cytomegalovirus infection J Virol 67:3166-75; Von Laer et al. (1995) Detection of cytomegalovirus DNA in CD34+ cells from blood and bone marrow Blood 86:4086-90). Viral genomes can be found in CD34+ cells from seropositive individuals and granulocyte-macrophage progenitors and differentiated macrophages can be infected experimentally (Soderberg et al. (1993) Definition of a subset of human peripheral blood mononuclear cells that are permissive to human cytomegalovirus infection J Virol 67:3166-75; Soderberg-Naucler et al. (1997) Reactivation of latent human cytomegalovirus by allogeneic stimulation of blood cells from healthy donors Cell 91:119-26). Furthermore, hematopoietic stem cells are also reported as a site for HCMV latency.
While the general feasibility of replication defective viral vectors for other cell types has been shown using other herpesviruses, e.g. HSV, EBV, and HHV-7 (Geller et al. (1997) Helper virus-free herpes simplex virus-1 plasmid vectors for gene therapy of Parkinson's disease and other neurological disorders Exp Neurol 144:98-102; Ho (1994) Amplicon-based herpes simplex virus vectors Methods Cell Biol 43:191-210; Jacobs et al. (1999) HSV-1-based vectors for gene therapy of neurological diseases and brain tumors: part II. Vector systems and applications Neoplasia 1:402-416; Kwong & Frenkel (1995) Biology of herpes simplex virus (HSV) defective viruses and development of the amplicon system Viral Vectors p. 25-42, Academic Press, Inc.; Mahmood et al. (1999) The role of HSV amplicon vectors in cancer gene therapy Gene Therapy and Molecular Biology 4:209-219; Romi et al. (1999) Tamplicon-7, a novel T-lymphotrpic vector derived from human herpesvirus 7 J Virol 73:7001-7007; Wang et al. (1998) Immune modulation of human B lymphocytes by gene transfer with recombinant Epstein-Barr virus amplicons J Virol Methods 72:81-93; Wang & Vos (1996) A hybrid herpesvirus infectious vector based on Epstein-Barr virus and herpes simplex virus type 1 for gene transfer into human cells in vitro and in vivo J Virol 70:8422-8430), efficient transduction of human CD34+ cells with retroviral and non-viral vectors has been unsatisfactory due to the lack of maintenance of high levels of expression of the transgene following engraftment of the engineered cells (Douglas et al. (2001) Efficient human immunodeficiency virus-based vector transduction of unstimulated human mobilized peripheral blood CD34+ cells in the SCID− hu Thy/Liv model of human Tcell lymphopoiesis Hum Gene Ther 12:401-13). The approaches to improving the efficiency of gene transfer into human cells have focused on improving gene delivery vectors and optimizing ex vivo culture conditions, which preserve the developmental properties of the stem cells (De Wynter et al. (1999) Properties of peripheral blood and cord blood stem cells Baillieres Best Pract Res Clin Haematol 12:1-17; Goerner et al. (2000) Expansion and transduction of nonenriched human cord blood cells using HS-5 conditioned medium and FLT3-L J Hematother Stem Cell Res 9:759-65).
The present invention exploits the tropism of HCMV to provide HCMV virus vectors which offer solutions to these and other problems.